1. Field of the Invention
This invention relates to electrophoresis systems and, more particularly, to novel ultra-thin, miniature, disposable, slab gel systems for speedy high resolution DNA analysis.
2. Discussion of Related Art
Electrophoresis is the resolution of a mixture of macromolecules on the basis of charge and/or size under the influence of an electric field. It is a primary analytical tool in molecular biology and biochemistry, used to separate mixtures of molecules such as proteins or nucleic acids in order to reveal their individual components. Electrophoretic analysis is based upon the fact that each molecule is characterized by a particular electrophoretic mobility under a given set of conditions. Polyacrylamide gel, agarose, cellulose or granulated gels are commonly used as stabilizing media, these gels being porous materials through which the sample components can migrate in an electric field. The electric field applied to the gel causes samples to migrate through the gel.
Deoxyribonucleic acid (DNA), for example, which is negatively charged, moves toward the positive electrode; smaller fragments of DNA migrating faster through the gel than larger fragments. DNA bands, consisting of resolved DNA chain sizes, can be detected by a number of methods. For example, isotopically labeled DNA may be detected by exposure of the gel to x-ray film.
DNA analysis can be separated into two categories: low resolution DNA analysis and high resolution DNA analysis. In low resolution analysis, double-stranded DNA fragments are run on non-denaturing polyacrylamide or agarose gels. The resolution obtained for such analysis is typically about plus or minus 10 or more nucleotide pairs. In high resolution analysis, single-stranded DNA fragments are run on denaturing polyacrylamide gels. This type of analysis separates DNA at single base resolution and is often used for DNA sequencing. The focus of the present invention is upon high resolution DNA analysis, although gel systems of the present invention are useful for either application.
It is common practice to conduct polyacrylamide gel electrophoresis in a buffered gel that was polymerized between two flat plates, usually transparent glass separated by plastic spacers. In order to provide accurate sample resolution, it is necessary that the gel thickness be uniform. It is important to avoid factors which affect electrophoretic mobility other than the characteristics of the molecules being separated. In traditional use, the gel is positioned vertically between two buffer chambers so that buffer is in contact with the gel. Samples are applied into wells formed at the top of the gel and a voltage is applied between the buffers which causes the samples to migrate within the gel. Upon completion of sample separation, the gel is separated from the plates for analysis.
Procedures for preparing and running gels for high resolution DNA analysis are presently hindered by several limitations. For instance, making and using conventional gels are technically demanding and labor intensive tasks, requiring substantial expertise. Additionally, a typical gel requires a relatively large DNA sample and must run for at least several hours to achieve adequate band separation. Further, glass plates used to make gels under current practices must be scrupulously cleaned before they can be reused.
Although the development of automated laser fluorescence systems represented a major breakthrough in large-scale DNA analysis, and some research institutions now have DNA sequencing facilities, many molecular biology laboratories still perform small-scale DNA analysis manually. For example, DNA sequencing gels, prepared in the laboratory, are often run with only a few samples loaded in order to verify or characterize a recombinant DNA construct. Also, there are other types of commonly-performed protocols, using isotope-labeled DNA, where the researcher wants to be able to quickly and easily analyze the DNA by denaturing gel electrophoresis in his or her own laboratory.
Attempts to increase the speed of electrophoretic separations have resulted in the development of capillary gel electrophoresis and ultrathin slab gels. In both of these methods, the lower electrical conductivity and greater heat dissipation than conventional gels allow much higher electric fields to be applied without damage to the gel by Joule heating. Electrophoresis times have been shortened approximately 20-fold compared with conventional methods. However, the long fused silica capillaries are expensive and difficult to work with, and ultrathin slab gels require special apparatus and techniques for pouring the gels.
Apparently, miniaturization had not been considered an option for DNA sequencing because it was generally beleived that small gels would lack the required resolution. This belief turns out to be incorrect when small volumes of sample are suitably loaded. At this time, the present inventors know of only one other miniaturized DNA sequencing system (Woolley, A. T. and Mathies, R. A., Anal. Chem. 1995, 67, 3676-3680). It was shown therein that DNA can be sequenced using gels formed in capillaries etched into small glass plates, termed capillary electrophoreseis (CE) chips. In the present invention, etched plates are not required. Each of the capillaries on a CE chip required 4 reservoirs with attached wires, two reservoirs for electrophoretic sample injection and two for sample separation. Reservoirs (60 in all) had to be sealed into the "chip" in a leak-free fashion. In the present invention, a novel, much simpler sample application system is used. Additionally, the CE chips required 1 .mu.l of a 100-fold concentrated sample (compared to a conventional sample) per lane. The high DNA concentration used was necessary in order to detect the fluorescently labeled DNA by laser confocal microscopy. This amount of DNA loaded corresponds to about 50 times more than is used for a traditional gel. In contrast, the present invention requires only about 1/20 the amount of DNA that is usually applied to a traditional gel.
The problems in the art are overcome by the present invention in which a simple inexpensive, disposable, miniaturized gel system is provided. In a preferred use, gel systems of the present invention are capable of very fast high resolution DNA analysis using extremely small samples.